Toxys is a Dutch biotech company that provides innovative in vitro toxicity screening solutions to rapidly identify hazardous and potential carcinogenic properties of novel compounds during the early phases of product and drug development.


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Toxys is developing the ReproTracker® assay, a state-of-the-art in vitro assay to visualize the key events during early embryonic development that allows us to reliably assess the potential developmental toxicity hazards of new drugs and products. This unique assay provides insight into the toxic mode-of-action.

Key Characteristics

  • Biomarker assay for developmental toxicity
  • Visualisation of key developmental pathways
  • Insight into mode of action
  • Human stem cell based assay

Biomarker based assay for developmental biology

Toxys is developing the ReproTracker® assay, a state-of-the-art in vitro assay to detect potential developmental toxicity hazards of chemical substances. The assay visualises the key events during early embryonic development which will allow us to quantify the progression of differentiation. We have combined our expertise on biomarker discovery and stem cell differentiation to design our new assay. ReproTracker® is able to identify compounds that interfere with the normal embryonic development and will provide insight into the underlying mechanisms of toxicity.

What is ReproTracker

Stem cells are widely accepted as an excellent platform for in vitro developmental toxicity hazard identification. ReproTracker® is a human stem cell-based assay, which uses a specific set of biomarkers to monitor the various stages of early embryonic development.

The selected biomarkers are chosen to represent specific phases of differentiation for both endoderm and mesoderm differentiation. When the pluripotent stem cells progress through the different stages of differentiation, the promoter of the selected marker genes will be activated. Should a compound interfere with the development, the biomarker will not be activated or activated at a different time. A considerable advantage to use this set of biomarkers, it is possible to see in one assay if a chemical can induce multiple teratogenic mechanisms. The response of the biomarkers to compounds will be quantified using qPCR.


Figure 1. Directed differentiation of matured hepatocytes. Pluripotent hiPSC were differentiated towards hepatocyte cells using Toxys protocol. Albumin IHC staining confirmed the presence of the hepatocyte-specific protein in the differentiated cells. Endoderm and hepatocyte-specific biomarker expression were followed by qPCR for 21 days. During differentiation, expression of the pluripotency marker OCT4 decreased while early endodermal biomarker expression (FoxA2and Sox17) was increased starting on day 3 and up-regulation of the matured liver-specific biomarker ALB was identified at day 21. 


Figure 2. Directed differentiation to matured cardiomyocytes. Pluripotent hiPSC were differentiated towards cardiomyocyte cells using Toxys protocol. TNNT2 IHC staining confirmed the presence of the cardiomyocyte-specific protein in the differentiated cells. Mesoderm and Cardiomyocyte specific biomarker expression was followed by qPCR for 14 days. During differentiation, expression of the pluripotency marker OCT4 decreased while early mesodermal biomarker expression (BMP4) was increased starting on day 3 and up-regulation of the matured cardiomyocyte-specific biomarker MYH6 was started increase from day 7. 


Pluripotency marker

POU Class 5 Homeobox 1 (POU5F1, aka OCT4) plays a key role in embryonic development and stem cell pluripotency. POU5F1/OCT4 is activated in pluripotent stem cells and works together with Sox2 and Nanog to suppress genes for differentiation and activate genes for self-renewal (Boyer et al., 2005). In ReproTracker® we are using the POU5F1 marker to detect the beginning of the differentiation program and the loss of pluripotency.

Early differentiation markers for endoderm lineage

SRY-box17 (SOX17) modulates the transcriptional regulation of Wnt signalling and inhibits the expression of pluripotency markers Sox2, Nanog and Oct4 (Niakan et al., 2010). Sox17 is highly expressed during early endoderm differentiation and required for formation of definitive endoderm that allows the formation of various organs, including the liver (Park et al. 2006). Sox 17 is one of the earliest biomarkers to detect early endodermal commitment.

FOXA2 hepatocyte nuclear factors are transcriptional activators for liver-specific genes such as albumin and transthyretinbinds (is there more than one FOXA2?). It binds to the Wnt6 promoter and together with Gata6, activates the Wnt/β-catenin pathway. This results in the formation of the extra-embryonic endoderm (Hwang & Kelly, 2012). Similarly to SOX17 reporters, FOXA2 used to detect differentiation towards the endodermal lineage. 

Early differentiation markers for mesoderm lineage

Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family,  which plays an essential role in embryonic development not only by enhancing self-renewal but also in directing their differentiation into the mesodermal lineage. We use BMP4 to identify the epithelial-mesenchymal transition via SLUG and MSX2, which further enhances mesodermal commitment (Richter et al. 2013).

Matured tissue markers

Albumin (ALB) waschosen to identify mature hepatocytes. Albumin is the most abundant serum protein produced by matured hepatocytes. Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains colloid osmotic pressure, in a blood vessel’s plasma. Cardiac muscle myosin is a hexamer consisting of two heavy chain subunits, two light chain subunits, and two regulatory subunits. We selected Myosin heavy chain 6 (MYH6)  gene as a biomarker for matured cardiomyocyte which encodes the alpha heavy chain subunit of cardiac myosin.



We are currently validating the assay using ECVAM reference compounds for Development And Reproductive Toxicants (DART).


The assay is currently under development, and therefore is not available for testing of novel compounds. Should you wish to stay updated on developments, or if you have reference compounds that you would like to test in the assays please contact us via the enquiries form below or by sending an email to

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